Discovery of SARS-CoV-2 papain-like protease (PLpro) inhibitors with efficacy in a murine infection model

Vaccines and first-generation antiviral therapeutics have provided important protection against COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, there remains a need for additional therapeutic options that provide enhanced efficacy and protection against potential viral resistance. The SARS-CoV-2 papain-like protease (PLpro) is one of the two essential cysteine proteases involved in viral replication. While inhibitors of the SARS-CoV-2 main protease have demonstrated clinical efficacy, known PLpro inhibitors have, to date, lacked the inhibitory potency and requisite pharmacokinetics to demonstrate that targeting PLpro translates to in vivo efficacy in a preclinical setting. Here, we report the machine learning–driven discovery of potent, selective, and orally available SARS-CoV-2 PLpro inhibitors, with lead compound PF-07957472 (4) providing robust efficacy in a mouse-adapted model of COVID-19 infection.

It is important to note that eq. 3 assumes a different PK-PD relationship to the trough-based efficacy relationship assumed for this project, requiring maintaining the unbound C min above the antiviral EC 90 .However, we view eq. 3 as a generally useful score applicable to compound design if careful consideration is made to limitations of the C avg,ss,unbound based approach on a given project.

Synthetically Accessible Bioisostere Replacement
One of the technologies used for machine learning driven parallel library design was our Synthetically Accessible Bioisostere Replacement (SABER) machine learning tool.SABER incorporates a model of bioactivity similarity that identifies bioisosteres for a given building block in parallel libraries.The model is built on activity data from a mixture of publicly available (33) and proprietary data, building on a fragmentation method for prediction similar to that in past neural networks for R-group replacement (34), as well as custom adaptations.
In addition to predicting the likelihood of a building block maintaining activity against a target once reacted, SABER has custom-built functionality to only deliver building blocks that meet the synthetic requirements of the library, and will not interfere with the reaction chemistry.The reaction requirements are delivered to SABER automatically when the user designs (see figure below) a library with the given reaction, and at least one monomer template for each leaf node of the library tree.SABER can then be selected to run on any given monomer of a specific leaf node.
A diagram showing the user interaction with SABER.A user draws the parallel library with the chemistry of interest using a graphical user interface and then is given the option to choose on which node to run SABER to find replacements for the query building block.
SABER can also work on more complicated libraries of varying depth and shape.It will also understand the synthetic requirements of these more complicated libraries including the necessary protecting groups that need to be present as shown in Figure 2.

Setting Up the Collaboration Workflow and Operations
Operationally, we designed the campaign to be resilient during a pandemic: to ensure rapid design-iteration and data feedback, we set up a robust logistical workflow using the global network of CROs, and existing Pfizer internal infrastructure.Biochemical and Tier 1/2 ADME assays were replicated externally and co-located with synthesis at an external CRO, while compounds were shipped immediately post-synthesis to a BSL3 level facility for antiviral assays.This setup also ensured compounds could efficiently enter gating viral assays postregistration, and quickly progress to further characterization.The exigencies of a pandemic showcase that the decentralized drug discovery model can be an effective way to achieve operational resilience and business continuity.

Preclinical Pharmacokinetics Studies
All activities involving animals were carried out in accordance with federal, state, local and institutional guidelines governing the use of laboratory animals in research in AAALAC accredited facilities and were reviewed and approved by Pfizer's or Bioduro's Institutional Animal Care and Use committee.

Mouse Pharmacokinetics
Mouse pharmacokinetic studies were done at BioDuro Pharmaceutical Product Development Inc. (Shanghai, PRC); male C57BL6 mice were purchased from Vital River (Beijing, China) and were typically 7-9 weeks of age at the time of dosing.During the pharmacokinetic studies all animals were housed individually.Access to food and water was provided ad libitum (i.e., subjects were dosed in the fed state).Compounds were administered intravenously (iv) via tail vein (n=2) dosed as a solution (1 mg/kg, 3 mL/kg) or via oral gavage as a solution (10 mg/kg, 3 mL/kg and 30 mg/kg, 3mL/kg) or as a suspension (15 mg/kg, 10 mL/kg; 50 mg/kg, 10 mL/kg; and 150 mg/kg, 10mL/kg).Dosing solutions were prepared immediately before dosing.The composition of each dosing vehicle is provided in Tables S5 and S6.Isoflurane was administered as anesthesia at 1-2% dose prior to collecting blood samples.Serial blood samples were collected via the retroorbital capillary at predetermined timepoints after dosing.Animals were monitored for pain or distress throughout the study, with at least daily monitoring during normal husbandry prior to study start.At the completion of the study, animals were euthanized by overdose of inhaled carbon dioxide.Blood samples were collected into tubes containing K 2 EDTA and stored on ice until centrifugation to obtain plasma, which was stored frozen at -20 °C or lower until bioanalysis.

Rat Pharmacokinetics
Rat pharmacokinetic studies were done at BioDuro Pharmaceutical Product Development Inc. (Shanghai, PRC); jugular vein-cannulated male Wistar-Hannover rats were purchased from Vital River (Beijing, China) and were typically 7-9 weeks of age at the time of dosing.During the pharmacokinetic studies all animals were housed individually.Access to food and water was provided ad libitum (i.e., subjects were dosed in the fed state).In the instances where oral (po) dose was administered in the fed state, with ad libitum access to food and water.Compounds were administered intravenously (iv) via the tail vein (n = 2) dosed as a solution (1 mg/kg, 3 ml/kg) or via oral gavage as a solution (10 mg/kg, 10 ml/kg).Dosing solutions were prepared immediately before dosing.The composition of each dosing vehicle is provided in Table S5.Serial blood samples were collected via the jugular vein cannula at predetermined timepoints after dosing.Animals were monitored for pain or distress throughout the study, with at least daily monitoring during normal husbandry prior to study start.At the completion of the study, animals were euthanized by overdose of inhaled carbon dioxide.Blood samples were collected into tubes containing K2EDTA and stored on ice until centrifugation to obtain plasma, which was stored frozen at -20 °C or lower.

Dog Pharmacokinetics
Dog pharmacokinetic studies were done at Pfizer (Groton, CT).All procedures performed on beagle dogs were in accordance with regulations and established guidelines and were reviewed and approved by an Institutional Animal Care and Use Committee through an ethical review process.Male Beagle dogs were purchased from Marshall Bioresources (North Rose, NY); subjects 3 years of age were used in pharmacokinetics studies.For each study (n=2), compounds were dosed iv by cephalic vein as a solution (0.5 mg/kg, 1 mL/kg) or via oral gavage as a suspension (3 mg/kg, 5 mL/kg).In the instances where oral (po) dose was administered in the fed state, dogs were fed a couple hours before dosing.In the instances where the IV dose was administered in the fasted state, dogs were fasted over night and fed 4 hours post dose.Subjects were monitored for pain or distress throughout the study followed by at least daily monitoring while off study.The iv dosing vehicle was optimized such that the compounds were in solution and stable for at least 24 h.Serial blood samples were collected via the jugular vein at predefined time points post-dose.Blood samples were collected into K 3 EDTA treated collection tubes and were stored on wet ice prior to being centrifuged to obtain plasma, which was stored frozen at -20 °C or lower.

Monkey Pharmacokinetics
Monkey pharmacokinetic studies were conducted at Pfizer (Groton, CT).All procedures performed on Cynomolgus monkeys were in accordance with regulations and established guidelines and were reviewed and approved by an Institutional Animal Care and Use Committee through an ethical review process.Male Cynomolgus monkeys were purchased from Envigo Global Services (Indianapolis, IN); subjects 5-8 years of age were used in pharmacokinetic studies.For each study (n=2), compounds were dosed iv by the cephalic vein (typically 1 mg/kg, 2 ml/kg).Subjects were monitored for pain or distress throughout the study followed by at least daily monitoring while off study.The iv dosing vehicle was optimized such that the compounds were in solution and stable for at least 24 h.Serial blood samples were collected via the femoral vein at predefined time points postdose.Blood samples were collected into K 3 EDTA treated collection tubes and were stored on wet ice prior to being centrifuged to obtain plasma, which was stored frozen at -20 °C or lower.

LC-MS/MS Analysis of Plasma Samples
Plasma samples were processed using protein precipitation with 50:50 acetonitrile: methanol containing terfenadine (5 ng/ml) as an internal standard followed by quantitation against a standard curve (0.1-1000 ng/ml) prepared in blank plasma.Quantitation of analyte in plasma samples was done using LC-MS/MS.Standard and quality control samples, prepared in blank plasma were extracted in the same manner as the in-life samples.Briefly, a Waters ACQUITY ultra performance liquid chromatography system (Waters, Milford, MA) coupled to an AB Sciex 5500 mass spectrometer equipped with an electrospray ionization source was used.Chromatographic separation was accomplished using a Kinetex C18 100A (2.6 µm, 3.00 × 50 mm) column maintained at room temperature.The mobile phase (2 solvent gradient) was optimized to achieve good separation between the analytes.Typically, solvent A consisted of 0.1% formic acid and 5 mM ammonium acetate in water, and solvent B included 0.1% formic acid and acetonitrile.The gradient generally began at 15% B until about 1.8 min, followed by an increase to 95% B to 2.21 min, then decreased to 15% B until 3 min.Analyst 1.6.1 software was used for peak integration and standard curve regression.

Pharmacokinetic Analysis
Pharmacokinetic parameters were calculated using noncompartmental analysis (Watson v.7.5, Thermo Scientific).The area under the plasma concentration-time curve from t = 0 to infinity (AUC 0-∞ ) was estimated using the linear trapezoidal rule.Plasma clearance (CL p ) was calculated as: The terminal rate constant (k el ) was calculated by linear regression of the terminal phase of the log-linear concentration-time curve and the terminal elimination t 1/2 was calculated as:  ' = 0.693 (  )* Apparent steady state distribution volume (Vd ss ) was determined by clearance multiplied by mean residence time.Oral bioavailability (F) was defined as:

Plasma Protein Binding Determination for PF-07957472
On the day of each incubation, fresh blood in K2EDTA was collected from male Wistar-Hannover rats (n=5, pooled), male Cynomolgus monkeys (n=2, pooled), and humans (n=1 male and n=1 female, pooled).The blood was centrifuged for 10 min at 2500 x g and the plasma fraction was harvested.Plasma was spiked with a final concentration of 0.3, 1, 3, or 10 µM of PF-07957472 (4) (final organic 1% DMSO).Fraction unbound in plasma (f u,p ) was determined by equilibrium dialysis using an HTD 96 device assembled with 12-14k molecular weight cutoff membranes (HTDialysis, LLC, Gales Ferry, CT).Dialysis chambers were loaded with 150 µl plasma and 150 µl PBS in the donor and receiver chambers, respectively.The dialysis plate was sealed with a gas-permeable membrane and stored in a 37 °C water-jacketed incubator maintained at 75% relative humidity and 5% CO 2 , on a 100 rpm plate shaker.After a 6-hour incubation, samples were matrix-matched and quenched by protein precipitation, followed by LC-MS/MS analysis (see LC-MS/MS Analysis of Plasma Samples section above).A set of satellite samples was included to measure stability after a 6-hour incubation.Incubations were conducted with 12 replicates per concentration.F u,p was calculated by dividing the analyte concentration in the buffer sample by the signal in the donor sample, corrected for any dilution factors.All incubations had >70% analyte recovery and >70% stability in 6 h.

CYP Inhibition Assay for PF-07957472
The effects of PF-07957472 on the CYP-mediated metabolism of selective probe substrates were characterized using human liver microsomes (pool of 50 male and female donors, purchased from XenoTech (Kansas City, Kansas) (37) ).The final solvent concentration in the incubation was <1%.Reactions were terminated after 6 minutes by quenching 175-µl of incubation mixture into 200 µl of acetonitrile containing a cassette of internal standards (a stable labeled internal standard that was specific to each analyte was used).Following vortex mixing and centrifugation at 2300 x g for 5 minutes, the resulting supernatants (350 µl) were transferred to clean 96-well plates, evaporated under a stream of warm nitrogen, and reconstituted in 100 µl of 90/10 water/acetonitrile followed by LC-MS/MS analysis.LC-MS/MS methods and instrumentation have been previously described.

Off Target Pharmacology Mammalian Protease Panel
These standard assays were run at Reaction Biology Corporation, except for cathepsin F at BPS Bioscience.

Coronavirus Protease Panel
A set of compounds was tested against a Coronavirus protease panel (SARS-CoV-1, SARS-CoV-2, 229E, MERS, and OC43), as described in the "PLPro Enzymatic Assay" section above, to determine their ability to inhibit each enzyme's activity.(7,15,16,(38)(39)(40) The potency of compounds against each protease was measured using a synthetic profluorogenic substrate, Z-RLRGG-AMC (GenScript).Compounds were serially diluted over 11 points using 100% DMSO as a diluent, from a top concentration of 1 mM with a half-log dilution factor.The final top dose of compounds in the assay was 10 µM (1% DMSO).The assay buffer contained 50 mM HEPES, pH 7.

Data Analysis for Mammalian and Coronavirus Protease Panels
Data were analyzed with ActivityBase software (IDBS).The no compound, zero percent inhibition (ZPE) control wells contained 1% DMSO with substrate and enzyme.The hundred percent effect (HPE) wells contained a control compound at a dose sufficient to accomplish complete inhibition (1% DMSO), substrate and enzyme.The raw data were transformed to percent activity values using the average from the ZPE and HPE control wells.The resulting data were fit with the four-parameter logistic fit model to determine the IC 50 value.Ki values were fit to the tight binding Morrison equation with fixed parameters for enzyme concentration, substrate concentration and the Km parameter using ActivityBase software (IDBS).

Compound Preparation
Compounds were dissolved and initially diluted in dimethyl sulfoxide (DMSO), with a final dilution in external solution to generate final working concentrations.The final DMSO concentration in all experiments was 0.33% (v/v).All tissue culture media and reagents were obtained from Thermo Fisher (Waltham, MA, USA), unless otherwise stated.Ion Channel experiments were performed at Metrion Biosciences Ltd (Cambridge, UK).
Ionic currents were evaluated in the whole-cell configuration using the Qube384 automated planar patch clamp platform (Sophion Bioscience A/S, Ballerup, Denmark).QChip 384X plates, containing 10 patch clamp holes per well, were used to maximize success rate, which was routinely > 95%.For hERG experiments, the external solution was composed of (in mM): 140 NaCl, 2 KCl, 2 CaCl 2 , 1 MgCl 2 , 10 HEPES, 5 Glucose, pH 7.4, 350 mOsM.The internal solution contained (in mM): 20 KCl, 10 EGTA, 10 HEPES, 120 KF, pH 7.2, 335 mOsM.The hERG current was elicited from a holding potential of -80 mV by a voltage step to +40 mV for 500 ms, followed by a repolarizing ramp to -80 mV at -0.6 mV/ms.This pattern was repeated at a rate of 0.05 Hz.Peak hERG current was measured during the ramp.All studies were conducted at 23 °C.
Three vehicle periods each lasting 5 minutes were applied to establish a stable baseline.This was followed by the addition of increasing concentrations of test compound, with each exposure lasting 5 minutes.Patch clamp data were analyzed using Analyzer Software 6.
Ionic currents were evaluated in the whole-cell configuration using the Qube384 automated planar patch clamp platform using QChip 384X plates.For hCav1.2 experiments, the external solution was composed of (in mM): 137.9 NaCl, 5.

X-ray Crystallography
SARS-CoV-2 PL pro protein purification and co-crystallization with PF-07957472 The codon-optimized gene corresponding to SARS-CoV-2 PL pro domain (Nsp3 residues 1562-1878; Uniprot P0DTD1; PL pro domain numbering 1-315) bearing a cysteine to serine mutation in the catalytic triad (C111S) was synthesized as a gBlock gene fragment by Integrated DNA Technologies and was subcloned into a pET28a vector using NcoI and XhoI restriction enzymes (New England Biolabs).The protein was expressed with a cleavable Nterminal His 6x tag in BL21(DE3) E. coli cells (Novagen Singles TM ).The protein was isolated from the lysate using Ni-NTA Agarose resin (Qiagen), the His 6x tag was cleaved using tobacco etch virus protease (TEV), and the tag-free construct was purified to homogeneity and >95% purity by size exclusion chromatography using a HiLoad Superdex 75 pg 16/600 column (Cytiva) in a buffer containing 50 mM HEPES pH 8, 200 mM NaCl, and 1 mM TCEP.
After concentrating the protein to 10 mg/mL, PF-07957472 was added to the protein at a final concentration of 300 μM.Crystals of the protein-ligand complex were obtained in a crystallization condition containing 0.1 M Sodium citrate tribasic dihydrate pH 5.0, 30% v/v Jeffamine® ED-2001 pH 7.0.
Diffraction Data, Structure Determination and Refinement X-ray crystal diffraction data were collected on the IMCA 17-ID beamlines (Advanced Photo Source, Argonne National Laboratory) using a wavelength of 0.95374 Å.All datasets were processed with autoPROC (Global Phasing).( 41) Anisotropy was corrected by ellipsoidal truncation using STARANISO as a part of autoPROC (Global Phasing) (42).Phasing for each structure was obtained by molecular replacement in PHASER ( 43) using as search model 7CJM.The crystals obtained of SARS-CoV-2 PL pro in complex with PF-07957472 were in the P212121 space group with three protomers in the asymmetric unit.Inhibitor density was strongest in two PL pro protomers, likely owing to crystal packing at the compound binding site.Trifluoroacetate was observed in the crystal packing interface, between chains A and B, which was present in the compound.Manual model building was performed in COOT (44) and refinement using BUSTER (Global Phasing) (45).Molecular graphics were generated using PyMOL (46).Data collection and refinement statistics are provided in Table S2.(500/500 mL).The organic layer was separated and the aqueous layer was re-extracted with ethyl acetate (500 ml).The combined organic layer was washed with brine, dried and evaporated in vacuo to give a residue.The residue was dissolved in 1,4-dioxane (500 mL) containing conc.HCl (50 mL) at 0 °C.The mixture was stirred at 20 °C for 15 h before being evaporated in vacuo to afford a crude.The crude was dissolved in 200 mL methanol and basified with ammonium hydroxide to pH = 9 at 0 °C.The mixture was then evaporated in vacuo and purified by silica gel chromatography using a Biotage (330 g column, petroleum ether/ethyl acetate = 1/0 to 0/1) to afford 1-(quinolin-4-yl)ethan-1-one (14850 mg, 90%) as a yellow oil.

Synthesis of 2-methyl-5-(4-methylpiperazin-1-yl)benzoic acid (int-1):
Step 1.A 500-mL 3-neck RBF equipped with overhead stirrer, internal temperature probe, and reflux condenser under nitrogen was charged with methyl 5-bromo-2-methylbenzoate (18 g, 78.6 mmol), cesium carbonate (76.8 g, 236 mmol), anhydrous dioxane (200 mL) and 1-methylpiperazine (9.6 mL, 86.4 mmol).The resulting yellow slurry was sparged with nitrogen under vigorous stirring for 15 min.The flask was briefly opened and RuPhosPdG 3 (3.3g, 3.93 mmol) was added in one portion and reaction mixture sparged for another 10 min.The reaction was heated under nitrogen in an aluminium block to 100 °C for 16 hours.The cooled reaction mixture was filtered over celite, rinsing with EA.The amber filtrate (600 mL total) was concentrated to remove dioxane, then dissolved in EA (150 mL).The EA solution was washed once with half-saturated ammonium chloride (75 mL).The aqueous was extracted once with EA (100 mL).The combined organics were extracted with 1M HCl (1 x 100 mL).The dark aq.layer was made neutral by the addition of 25 mL 4M NaOH, then extracted with EA (150 mL).TLC showed desired product still present in the aq.layer, so it was made basic (pH~12) by the addition of 4 mL 4M NaOH and extracted again with EA (100 mL).By TLC, almost all of the desired product was now in the combined organics.The combined organics were washed once with saturated brine, dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure to afford 17.98 g (92%) of methyl 2-methyl-5-( 4 Step 2. A solution of methyl 2-methyl-5-(4-methylpiperazin-1-yl)benzoate (17.98 g, 72.4 mmol) in EtOH (54 mL) and water (18 mL) was treated with lithium hydroxide monohydrate (3.1 g, 79.6 mmol) and the reaction mixture was heated to 60 °C for 16 hrs.The reaction was cooled to rt and the amber-colored solution was treated with con HCl gradually until pH = 3 (12 mL).A solid formed and the entire reaction became a solid block of beige clay.The solid was treated with 150 mL EtOH and broken up with a spatula until a thick, but stirrable, slurry formed.The beige slurry was stirred at rt for 1.5 hrs and then filtered, rinsing the filter cake

Figure S2 .
Figure S2.BL2 binding loop from human coronavirus papain-like proteases.(A) sequence alignment of the papain-like proteases from SARS-CoV-2, SARS-CoV-1, MERS, 229E, OC43, and BtSCoV-RF1.2004.The non-conserved amino acids are highlighted in red boxes, green/blue indicate sharing similarity while identical residues are kept in a grey background.(B) Structural comparison of BL2 loop of PL pro from SARS-CoV-2 (orange pdb ID this study) and MERS (cyan, pdb ID 4RF0) showing the high flexibility in the loop and sequence variability especially betweenTyr268/Gln269 from SARS-CoV that make direct interactions with compound 4 (green) and Glu273/Thr274 from MERS.(C) Full view of PL pro indicating

Table S7 :
Mouse multi-dose FormulationOral pharmacokinetic studies were conducted in the fed state unless otherwise noted.Oral rat pharmacokinetic studies were conducted with xx or xx form xx. The aforementioned lowdose 1 mg/kg IV was formulated as solutions in 10% DMSO / 90% (23% w/v) HPBCD in DI water (v/v).The 10 mg/kg PO doses are included for comparison.The For the 15, 50, 150 mg/kg doses, a homogeneous suspension formulation in 2% (v/v) Tween80 in 98% (v/v) of 0.5% (w/v) MethylCellulose by utilizing Geometric dilution were prepared.

Figure S3 -
Figure S3 -EC50.In vitro -in vivo -correlation of compound potency.The EC 50 (A) and EC 90 (B) were calculated for the compound in BalbC mice based on the projected Cmin and lung virus titer after 4 days of treatment (points = data, solid black line = fitted sigmoidal curve).The EC50 and EC 90 s calculated from Vero E6 (red) and HAE (blue) cells were overlayed and revealed that the
3 KCl, 10 HEPES, 0.49 MgCl 2 , 10 CaCl 2 , 5.5 glucose, 4.16 NaHCO 3 , 0.34 Na 2 HPO 4 , 0.41 MgSO Hz, and hCav1.2currentwasmeasuredas the peak current at 0 mV.All studies were conducted at 23 °C.Three vehicle periods each lasting 5 minutes were applied to establish a stable baseline.This was followed by the addition of increasing concentrations of test compound, with each exposure lasting 5 minutes.Patch clamp data were analyzed using Analyzer Software 6.4.72.Current amplitudes were determined by averaging the last 4 currents under each test condition.The percentage inhibition of each compound was determined by taking the ratio of current amplitude measured in the presence of various concentrations of the test compound (I Compound ) versus the vehicle control current (I Vehicle ): % Inhibition = [1-(I Compound /I Vehicle )] * 100%.A dose-response curve was generated and fit to the Hill equation by the Sophion Analyzer software to determine an IC 50 value for each compound.The minimum and the slope of the fit were free fitted, with the top being fixed to 100% inhibition.Hz, wit the hNav1.5 peak current defined as the maximum current during the step to -15 mV.All studies were conducted at 23 °C Three vehicle periods each lasting 5 minutes were applied to establish a stable baseline.This was followed by the addition of increasing concentrations of test compound, with each exposure lasting 5 minutes.Patch clamp data were analyzed using Analyzer Software 6.4.72.Current amplitudes were determined by averaging the last 4 currents under each test condition.The percentage inhibition of each compound was determined by taking the ratio of current amplitude measured in the presence of various concentrations of the test compound (I Compound ) versus the vehicle control current (I Vehicle ): % Inhibition = [1-(I Compound /I Vehicle )] * 100%.A dose-response curve was generated and fit to the Hill equation by the Sophion Analyzer software to determine an IC 50 value for each compound.The minimum and the slope of the fit were free fitted, with the top being fixed to 100% inhibition.CEREP in vitro pharmacology panel PF-07957472 ScreeningSecondary (off-target) pharmacology studies were conducted by Eurofins Cerep (Celle-Lévescault, France) on behalf of Pfizer, Inc.The in vitro off-target pharmacology of PF-07957472 was assessed at 30 µM in a broad target profiling panel which represents targets with known links to potential safety concerns and includes G-protein coupled receptors, ion channels, transporters, and enzymes according to established protocols.

Table S2 .
Data collection and refinement statistics.
All chemicals, reagents, and solvents were purchased from commercial sources and were used without further purification.1H NMR data are reported relative to residual solvent signals and are reported as follows: chemical shift (ppm), multiplicity, coupling constant (Hz), and integration.The multiplicities are denoted as: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br s, broad singlet.Silica gel chromatography was performed using Biotage or ISCO purification systems with pre-packaged columns.Concentration under reduced pressure was performed on a rotary evaporator with a water bath temperature not exceeding 60 C. Purity of final compounds was assessed by HPLC with UV detection at 215 or 254 nm; all tested compounds showed > 90% purity.
toluene (60.0 mL) under N 2 , was added diphenylphosphoryl azide (3060 mg, 11.1 mmol) and TEA (2250 mg, 22.2 mmol) at 20 °C.The reaction mixture was stirred at 90 °C under N 2 for 2 h, then evaporated in vacuo to afford a crude.The crude was dissolved in 1,4dioxane (40mL), followed by adding aq.NaOH (10%, 20 mL) at 20 °C under N 2 .The reaction mixture was stirred at 90 °C for 15 h.LCMS showed a mass peak of the desired product.The reaction mixture was then partitioned between ethyl acetate and H 2 O (250/150 mL).The organic layer was separated, and the aqueous layer was re-extracted with ethyl acetate (50 mL).The combined organic layer was washed with brine, dried with anhydrous Na 2 SO 4 , filtered and concentrated in vacuo.The residue was dissolved in 30 mL MeOH, then 10 mL 1 N HCl was added to the solution at 20 °C.The reaction mixture was stirred at 20 °C for 1 h, then partitioned between ethyl acetate and H 2 O (250/150 mL).The organic layer was discarded, and the aqueous layer was basified with NH 3 .H 2 O to pH = 9 at 0 °C, followed by extraction with DCM (200 mL).The organic layer was washed with brine, dried with anhydrous Na 2 SO 4 and concentrated in vacuo to afford 1-(2-chloroquinolin-4-yl)cyclopropan-1-amine (1444 mg, 65.4%) as a white solid.LCMS m/z calc.for C 12 H 12 ClN 2 + [M+H] + 219.1, found 219.1.

Table S5 .
Cytotoxicity testing of PF-07957472 in recombinant cell lines used for antiviral assays.Cytotoxicity of PF-07957472 was evaluated in non-infected Vero E6 enriched for ACE2.A P-glycoprotein inhibitor, CP-100356 was added at dose indicated to inhibit the efflux of PF-07957472 from Vero cells.Compound cytotoxicity was calculated as CC50 relative to DMSO treated and a cytotoxic control compound.

Table S6 .
Low-dose Preclinical pharmacokinetics of PF-07957472 a Pharmacokinetic parameters were calculated from plasma concentration-time data and are reported as mean (± S.D. for n=3 and individual values for n=2).All pharmacokinetics were conducted in male sex of each species.Oral (po) studies were conducted in the fed state unless otherwise noted.

Table S8 . Mutation count/ frequency of SARS-CoV-2 PLPro for key contact residues within 5A around PF-07957472 and the catalytic /tunnel residues.
The mutation annotation was performed on 6,806,275 Omicron genome sequences collected from GISAID as of 12/08/2023